1. Field of the Invention
The present invention relates generally to methods for generating antigen-specific transfer factor, compositions including such antigen-specific transfer factor, and uses of these compositions. In particular, the present invention relates to methods for generating antigen-specific transfer factor in an avian host and obtaining the antigen-specific transfer factor from eggs.
2. Background of Related Art
Many deadly pathogens are passed to humans from the animal kingdom. For example, monkeys are the sources of the type I human immunodeficiency virus (HIV-I), which causes acquired immune deficiency syndrome (AIDS) and monkeypox, which is similar to smallpox; ground-dwelling mammals are believed to be the source of the Ebola virus; fruit bats and pigs are the source of the Nipah virus; the Hendra virus comes from horses; the xe2x80x9cHong Kong Fluxe2x80x9d originated in chickens; and wild birds, especially ducks, are the sources of many of the deadly influenza viruses. Many diseases also have animal reservoirs. By way of example, mice carry Hanta virus, rats carry the Black Plague, and deer carry Lyme disease.
The immune systems of vertebrates are equipped to recognize and defend the body from invading pathogenic organisms, such as parasites, bacteria, fungi, and viruses. Vertebrate immune systems typically include a cellular component and a noncellular component.
The cellular component of an immune system includes the so-called lymphocytes, or white blood cells, of which there are several types. It is the cellular component of a mature immune system that typically mounts a primary, nonspecific response to invading pathogens, as well as being involved in a secondary, specific response to pathogens.
In the primary, or initial, response to an infection by a pathogen, white blood cells that are known as phagocytes locate and attack the invading pathogens. Typically, a phagocyte will internalize, or xe2x80x9ceatxe2x80x9d a pathogen, then digest the pathogen. In addition, white blood cells produce and excrete chemicals in response to pathogenic infections that are intended to attack the pathogens or assist in directing the attack on pathogens.
Only if an infection by invading pathogens continues to elude the primary immune response is a specific, secondary immune response to the pathogen needed. As this secondary immune response is typically delayed, it is also known as xe2x80x9cdelayed-type hypersensitivityxe2x80x9d. A mammal, on its own, will typically not elicit a secondary immune response to a pathogen until about seven (7) to about fourteen (14) days after becoming infected with the pathogen. The secondary immune response is also referred to as an acquired immunity to specific pathogens. Pathogens have one or more characteristic proteins, which are referred to as xe2x80x9cantigensxe2x80x9d. In a secondary immune response, white blood cells known as B lymphocytes, or xe2x80x9cB-cellsxe2x80x9d, and T lymphocytes, or xe2x80x9cT-cellsxe2x80x9d, xe2x80x9clearnxe2x80x9d to recognize one or more of the antigens of a pathogen. The B-cells and T-cells work together to generate proteins called xe2x80x9cantibodiesxe2x80x9d, which are specific for one or more certain antigens on a pathogen.
The T-cells are primarily responsible for the secondary, or delayed-type hypersensitivity, immune response to a pathogen or antigenic agent. There are three types of T-cells: T-helper cells, T-suppressor cells, and antigen-specific T-cells, which are also referred to as cytotoxic (meaning xe2x80x9ccell-killingxe2x80x9d) T-lymphocytes (xe2x80x9cCTLsxe2x80x9d), or T-killer cells. The T-helper and T-suppressor cells, while not specific for certain antigens, perform conditioning functions (e.g., the inflammation that typically accompanies an infection) that assist in the removal of pathogens or antigenic agents from an infected host.
Antibodies, which make up only a part of the noncellular component of an immune system, recognize specific antigens and, thus, are said to be xe2x80x9cantigen-specificxe2x80x9d. The generated antibodies then basically assist the white blood cells in locating and eliminating the pathogen from the body. Typically, once a white blood cell has generated an antibody against a pathogen, the white blood cell and all of its progenitors continue to produce the antibody. After an infection is eliminated, a small number of T-cells and B-cells that correspond to the recognized antigens are retained in a xe2x80x9crestingxe2x80x9d state. When the corresponding pathogenic or antigenic agents again infect the host, the xe2x80x9crestingxe2x80x9d T-cells and B-cells activate and, within about forty-eight (48) hours, induce a rapid immune response. By responding in this manner, the immune system mounts a secondary immune response to a pathogen, the immune system is said to have a xe2x80x9cmemoryxe2x80x9d for that pathogen.
Mammalian immune systems are also known to produce smaller proteins, known as xe2x80x9ctransfer factors,xe2x80x9d as part of a secondary immune response to infecting pathogens. Transfer factors are another noncellular part of a mammalian immune system. Antigen-specific transfer factors are believed to be structurally analogous to antibodies, but on a much smaller molecular scale. Both antigen-specific transfer factors and antibodies include antigen-specific cites and both include highly conserved regions that interact with receptor sites on their respective effector cells. In transfer factor and antibody molecules, a third, xe2x80x9clinkerxe2x80x9d, region connects the antigen-specific cites and the highly conserved regions.
Transfer factor is a low molecular weight isolate of lymphocytes. Narrowly, transfer factors may have specificity for single antigens. U.S. Pat. Nos. 5,840,700 and 5,470,835, both of which issued to Kirkpatrick et al. (hereinafter collectively referred to as xe2x80x9cthe Kirkpatrick Patentsxe2x80x9d), disclose the isolation of transfer factors that are specific for certain antigens. More broadly, xe2x80x9cspecificxe2x80x9d transfer factors have been generated from cell cultures of monoclonal lymphocytes. Even if these transfer factors are generated against a single pathogen, they have specificity for a variety of antigenic sites of that pathogen. Thus, these transfer factors are said to be xe2x80x9cpathogen-specificxe2x80x9d rather than antigen-specific. Similarly, transfer factors that are obtained from a host that has been infected with a certain pathogen are pathogen-specific. Although such preparations are often referred to in the art as being xe2x80x9cantigen-specificxe2x80x9d due to their ability to elicit a secondary immune response when a particular antigen is present, transfer factors having different specificities may also be present. Thus, even the so-called xe2x80x9cantigen-specificxe2x80x9d, pathogen-specific transfer factor preparations may be specific for a variety of antigens.
Additionally, it is believed that antigen-specific and pathogen-specific transfer factors may cause a host to elicit a delayed-type hypersensitivity immune response to pathogens or antigens for which such transfer factor molecules are not specific. Transfer factor xe2x80x9cdrawsxe2x80x9d at least the non-specific T-cells, the T-inducer and T-suppressor cells, to an infecting pathogen or antigenic agent to facilitate a secondary, or delayed-type hypersensitivity, immune response to the infecting pathogen or antigenic agent.
Typically, transfer factor includes an isolate of proteins obtained from immunologically active mammalian sources and having molecular weights of less than about 10,000 daltons (D). It is known that transfer factor, when added either in vitro or in vivo to mammalian immune cell systems, improves or normalizes the response of the recipient mammalian immune system.
The immune systems of newborns have typically not developed, or xe2x80x9cmaturedxe2x80x9d, enough to effectively defend the newborn from invading pathogens. Moreover, prior to birth, many mammals are protected from a wide range of pathogens by their mothers. Thus, many newborn mammals cannot immediately elicit a secondary response to a variety of pathogens. Rather, newborn mammals are typically given secondary immunity to pathogens by their mothers. One way in which mothers are known to boost the immune systems of newborns is by providing the newborn with a set of transfer factors. In mammals, transfer factor is provided by a mother to a newborn in colostrum, which is typically replaced by the mother""s milk after a day or two. Transfer factor basically transfers the mother""s acquired, specific (i.e., delayed-type hypersensitive) immunity to the newborn. This transferred immunity typically conditions the cells of the newborn""s immune system to react against pathogens in an antigen-specific manner, as well as in an antigen- or pathogen-nonspecific fashion, until the newborn""s immune system is able on its own to defend the newborn from pathogens. Thus, when transfer factor is present, the immune system of the newborn is conditioned to react to pathogens with a hypersensitive response, such as that which occurs with a typical delayed-type hypersensitivity response. Accordingly, transfer factor is said to xe2x80x9cjump startxe2x80x9d the responsiveness of immune systems to pathogens.
Much of the research involving transfer factor has been conducted in recent years. Currently, it is believed that transfer factor is a protein with a length of about forty-four (44) amino acids. Transfer factor is believed to have a molecular weight in the range of about 4,000 to about 5,000 Daltons (D), or about 4 kD to about 5 kD. Transfer factor is also believed to include three functional fractions: an inducer fraction; an immune suppressor fraction; and an antigen-specific fraction. Many in the art believe that transfer factor also includes a nucleoside portion, which could be connected to the protein molecule or separate therefrom, that may enhance the ability of transfer factor to cause a mammalian immune system to elicit a secondary immune response. The nucleoside portion may be part of the inducer or suppressor fractions of transfer factor.
The antigen-specific region of the antigen-specific transfer factors is believed to comprise about eight (8) to about twelve (12) amino acids. A second highly-conserved region of about ten (10) amino acids is thought to be a very high-affinity T-cell receptor binding region. The remaining amino acids may serve to link the two active regions or may have additional, as yet undiscovered properties. The antigen-specific region of a transfer factor molecule, which is analogous to the known antigen-specific structure of antibodies, but on a much smaller molecular weight scale, appears to be hyper-variable and is adapted to recognize a characteristic protein on one or more pathogens. The inducer and immune suppressor fractions are believed to impart transfer factor with its ability to condition the various cells of the immune system so that the cells are more fully responsive to the pathogenic stimuli in their environment.
Conventionally, transfer factor has been obtained from the colostrum of milk cows. While milk cows typically produce large amounts of colostrum and, thus, large amounts of transfer factor over a relatively short period of time, milk cows only produce colostrum for about a day or a day-and-a-half every year. Thus, milk cows are neither a constant source of transfer factor nor an efficient source of transfer factor.
Transfer factor has also been obtained from a wide variety of other mammalian sources. For example, in researching transfer factor, mice have been used as a source for transfer factor. Antigens are typically introduced subcutaneously into mice, which are then sacrificed following a delayed-type hypersensitivity reaction to the antigens. Transfer factor is then obtained from spleen cells of the mice.
While different mechanisms are typically used to generate the production of antibodies, the original source for antibodies may also be mammalian. For example, monoclonal antibodies may be obtained by injecting a mouse, rabbit, or other mammal with an antigen, obtaining antibody-producing cells from the mammal, then fusing the antibody-producing cells with immortalized cells to produce a hybridoma cell line, which will continue to produce the monoclonal antibodies throughout several generations of cells and, thus, for long periods of time.
Antibodies against mammalian pathogens have been obtained from a wide variety of sources, including mice, rabbits, pigs, cows, and other mammals. In addition, the pathogens that cause some human diseases, such as the common cold, are known to originate in birds. As it has become recognized that avian (i.e., bird) immune systems and mammalian immune systems are very similar, some researchers have turned to birds as a source for generating antibodies.
U.S. Pat. No. 5,080,895, issued to Tokoro on Jan. 14, 1992 (hereinafter xe2x80x9cthe ""895 Patentxe2x80x9d), discloses a method that includes injecting hens with pathogens that cause intestinal infectious diseases in neonatal mammals. The hens then produce antibodies that are specific for these pathogens, which are present in eggs laid by the hens. The ""895 Patent discloses compositions that include these pathogen-specific antibodies and use thereof to treat and prevent intestinal diseases in neonatal piglets and calves. In addition, the ""895 Patent assumes that a pathogen-specific transfer factor-like substance is passed from a hen to her eggs. Nonetheless, the ""895 Patent does not disclose that such a transfer factor-like substance was in fact present in the eggs, or that an antibody-free composition derived from eggs that were assumed to contain this transfer factor-like substance actually treated or prevented intestinal diseases in neonatal mammals. In fact, the ""895 Patent discloses the use of a filter with about 0.45 xcexcm diameter holes to isolate transfer factor from antibodies. As those of skill in the art are aware, however, antibodies, larger molecules, viruses, and even some bacteria will pass through the pores of a 0.45 xcexcm filter. In reality, it is not likely that any individual protein molecules having molecular weights of less than about 12,000 D were separated by such a filter. Based on the pore size of the filter used, however, it is more likely that no individual protein molecules, including antibodies, were removed by the filter.
Avian antibodies that are specific for mammalian pathogens have also been obtained by introducing antigens into eggs.
Treatment of pathogenic infections in mammals with avian antibodies is typically not desirable, however, since the immune systems of mammals are likely to respond negatively to the large avian antibody molecules by eliciting an immune response to the antibodies themselves. Moreover, as mammalian immune systems do not recognize avian antibodies as useful for their abilities to recognize certain pathogens, or the specificities of avian antibodies for antigens of such pathogens, avian antibodies do not even elicit the desired immune responses in mammals.
The inventors are not aware of any art that teaches a method for generating transfer factor in a non-mammalian source, an efficient method for obtaining transfer factor from such a non-mammalian source, such as an avian source, or a method for using such transfer factor in treating or preventing infections by pathogens.
The present invention includes a method for generating the production of transfer factor in a non-mammalian source and obtaining transfer factor from a non-mammalian source. In addition, compositions including non-mammalian transfer factor are also within the scope of the present invention, as are methods of using these compositions.
The non-mammalian transfer factor generated, obtained, and used in accordance with the present invention may either be antigen non-specific or antigen-specific (i.e., configured to bind or recognize one or more antigens). Unless otherwise indicated, the term xe2x80x9ctransfer factorxe2x80x9d, as used herein, includes the previously discussed broad definition, which includes each of the various types of transfer factors, including pathogen-specific, antigen-specific, and transfer factors that are not specific for particular pathogens or antigenic agents. The term xe2x80x9cnon-specificxe2x80x9d, when used herein with respect to transfer factors, refers to both transfer factors that are not specific for particular antigens and to mixtures that include transfer factors with different antigen specificities.
Non-specific transfer factor includes transfer factor that the non-mammalian source animal already produces. Individual non-specific transfer factor molecules that are produced by the source animal may have specificity for various antigenic agents, including pathogens, that are present in the source animal""s environment. Nonetheless, for purposes of the present invention, transfer factor that is generated merely by a source animal""s reaction to its environment is referred to as xe2x80x9cnon-specificxe2x80x9d.
On the other hand, antigen-specific transfer factor is generated by exposing a non-mammalian source animal to one or more antigens. The antigens of various types of pathogens, including, but not limited to, bacteria, viruses, fungi, and parasites, have been found by the inventors to induce the production of non-specific transfer factor in non-mammalian sources. Antigen-specific transfer factor has been generated by non-mammalian source animals by both natural antigens (including from live, inactivated, and attenuated sources) and synthetic antigens.
The production of transfer factor in a non-mammalian source may be induced by introducing an antigen characteristic of a certain pathogen into a female non-mammalian source animal. Exemplary types of source animals that may be used include, without limiting the scope of the present invention, birds, reptiles, amphibians, and fish. Preferably, the non-mammalian source animal produces eggs on a frequent basis. Thus, for purposes of the present invention, hens are particularly useful as the non-mammalian source animal. These non-mammalian source animals produce transfer factor, which then appears in the eggs of these source animals. Alternatively, an egg of a non-mammalian source animal may be exposed to the antigenic agent (e.g., by injection of the antigenic agent into the egg) to induce production of transfer factor by the egg itself.
The transfer factor generated by a non-mammalian source animal or by the egg of a non-mammalian source animal may be recovered from the egg and separated from other constituents of the egg, including proteins of larger molecular weight, such as antibodies. Alternatively, transfer factor may be purified from one or more eggs of a non-mammalian source animal.
The non-mammalian transfer factor may then be incorporated into a composition or apparatus for administration to a mammalian or non-mammalian subject or administered directly to the subject. The non-mammalian transfer factor or compositions including the non-mammalian transfer factor may be administered enterally (i.e., orally), or parenterally (i.e., by a non-oral route, such as by injection, through the skin, etc.). Administration of both non-specific and specific non-mammalian transfer factors have been found to initiate an early, specific (i.e., secondary) immune response in mammals to various invading pathogens. Thus, non-mammalian transfer factor has been found to be useful in treating and preventing diseases that may be caused by these various pathogens.
Other features and advantages of the present invention will become apparent to those of skill in the art through consideration of the ensuing description, the accompanying drawings, and the appended claims.